Respiratory diseases are a major cause of economic loss to the equine industry. Equine herpesviruses (EHV), equine influenza viruses (EIV), and the bacterium, Streptococcus equi are pathogens most often associated with infectious respiratory disease in horses. World wide, equine herpesviruses are major pathogens associated with morbidity in horses as a result of respiratory infection. Both equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) can cause respiratory disease. EHV-1 is also associated with abortions and neurological disease. Because of the high degree of mobility and the international nature of the equine industry, efficacious vaccines are needed to reduce the disease and control the spread of these pathogens.
A number of EHV vaccines are available commercially. None, however, generally is capable of conferring long lasting protection and most require frequent booster immunizations to achieve a significant level of protection against EHV infection. The most commonly recommended route of administration is via intramuscular injection, despite the respiratory system being a primary site of the infection in many instances. In addition, some of the commercial vaccines have been reported to cause undesirable side effects. A number of attempts at developing a recombinant vaccine for EHV have been reported. This approach, however, has not yet resulted in the introduction of a commercial recombinant vaccine which has achieved widespread acceptance.
Literature reports have consistently documented a high degree of variability in the capability of vaccines based on EHV-1 strains to provide cross protection against infection by EHV-4 strains. While vaccines based on EHV-4 strains have shown a greater propensity to provide some protection against both EHV-1 and EHV-4 strains, cross protection based on EHV-4 strains has also been reported to show variability.
There is accordingly a continuing need to develop additional vaccines capable of protecting horses against diseases associated with EHV-1 and/or EHV-4. It would also be advantageous to develop vaccine that is effective against EHV-1 and/or EHV-4 which could be administered via intranasally as well as via parenteral methods (e.g., intramuscularly, subcutaneously or intravenously).
The present invention relates to immunogenic compositions which include an inactivated form of EHV-1. In particular, the application provides a vaccine for protecting horses against diseases associated with EHV-1 and/or EHV-4. The vaccine includes inactivated EHV-1 (e.g., chemically inactivated EHV-1 KyA virus) and typically also includes an adjuvant. The vaccine may also include other components, such as preservative(s), stabilizer(s) and antigens against other equine pathogens. Typically, the antigens against other equine pathogens are also present in an inactivated form, such as inactivated forms of EHV-4 and inactivated strains of equine influenza virus (xe2x80x9cEIVxe2x80x9d). For example, the vaccine may be a combination vaccine which includes inactivated forms of A1 and/or A2 strains of equine influenza virus in addition to the inactivated EHV-1. Examples of suitable antigens against EIV include inactivated EIV A1 virus strain A/EQ1/Newmarket/77, inactivated EIV A2 virus strain Newmarket/2/93, and inactivated EIV A2 virus strain Kentucky/95.
The terms xe2x80x9cvaccinexe2x80x9d and xe2x80x9cimmunogenic compositionxe2x80x9d are defined herein in a broad sense to refer to any type of biological agent in an administratable form capable of stimulating an immune response in an animal inoculated with the vaccine. For purposes of this invention, the vaccine (immunogenic composition) typically includes the viral agent in an inactivated form. Vaccines in general may be based on either the virus itself or an immunogenic (antigenic) component of the virus. Herein, the term xe2x80x9cprotectionxe2x80x9d when used in reference to a vaccine refers to the amelioration (either partial or complete) of any of the symptoms associated with the disease or condition in question. Thus, protection of horses from EHV by the present vaccines generally results in a diminishing of virus shedding and/or one or more of the clinical symptoms associated with infection by EHV-1 and/or EHV-4 (e.g., pyrexia, nasal discharge, conjunctivitis, coughing, dyspnea, depression, and antibiotic treatment required for secondary bacterial infection).
In one embodiment, the present immunogenic compositions include a chemically inactivated form of EHV-1. Vaccines which include chemically inactivated EHV-1 KyA virus are particularly desirable. A variety of chemical inactivating agents known to those skilled in the art may be employed to inactivate the virus. Ethylenimine and related derivatives, such as binary ethylenimine (xe2x80x9cBEIxe2x80x9d) and acetylethylenimine, are examples of suitable chemical inactivating agents for use in inactivating the EHV-1 virus. Other chemical inactivating agents, e.g., beta-propiolactone or aldehydes (such as formaldehyde and glutaraldehyde), can also be used to inactivate the virus.
The present vaccines generally include an adjuvant which desirably may have bioadhesive properties, particularly where the virus is designed to be capable of intranasal administration. Examples of suitable adjuvants include cross-linked olefinically unsaturated carboxylic acid polymers, such as cross-linked acrylic acid polymers. As used herein the term xe2x80x9ccross-linked acrylic acid polymerxe2x80x9d refers to polymer and copolymers formed from a monomer mixture which includes acrylic acid as the preodominant monomer in the mixture. Examples of suitable cross-linked acrylic acid polymers include those commercially available under the tradenames Carbopol(copyright) 934P and Carbopol(copyright) 971 (available from B. F. Goodrich Co., Cleveland, Ohio). One particularly suitable adjuvant for use in the present vaccines is a cross-linked acrylic acid polymer having a Brookfield viscosity of no more than about 20,000 cPs (as measured at 20 rpm as a 1.0 wt. % aqueous solution at pH 7.5). Where a bioadhesive adjuvant is desired, it may be advantageous to utilize an adjuvant which has a bioadhesive property of at least about 50 dynes/cm2 as measured between two pieces of freshly excised rabbit stomach tissue (as determined by the procedure described in U.S. Pat. No. 4,615,697).
Methods for protecting horses against diseases associated with EHV-1 and/or EHV-4 which include administering a vaccine containing inactivated EHV-1 to the horses. The vaccine can be administered using a variety of methods including intranasal and/or parenteral (e.g., intramuscular) administration. In one embodiment of the method, the inactivated EHV-1 containing vaccine is first administered intramuscularly one or more times (e.g., at intervals of 2-4 weeks), followed by administration of the vaccine at least once intranasally (e.g., 2-4 weeks after the last parenteral administration of vaccine). The vaccine is advisedly administered to horses that are 6 months or older. Ideally, all horses in a given herd are vaccinated annually in order to protect against the spread of respiratory symptoms of the disease.
A method of producing an equine herpesvirus vaccine is also provided. The method typically includes inoculating simian cells with EHV-1 virus, e.g., with EHV-1 KyA virus. The inoculated simian cells are incubated, generally at least until CPE is observed (commonly after 24 to 120 hours at 36xc2x0 C.), and then the EHV-1 virus is harvested from the incubated cells (e.g., by decanting and filtering the culture fluids). The harvested virus-containing fluids can be treated with a chemical inactivating agent, such as binary ethylenimine, to form inactivated EHV-1 virus. Typically, the inactivated virus is further processed, e.g., by concentration and blending with other components, to produce a commercial formulation. For example, the fluids containing the inactivated virus may be concentrated and blended with an adjuvant and/or antigen(s) to one or more other equine pathogens.
The present application is also directed to a kit which includes in combination, (1) a dispenser capable of administering a vaccine to a horse; and (2) a chemically inactivated EHV-1 containing vaccine capable of protecting against diseases associated with EHV-1 and/or EHV-4. The kit may include a dispenser which is capable of dispensing its contents as droplets, e.g., as aerosol, atomized spray and/or liquid droplets, and a form of the vaccine which is capable of protecting against diseases associated with EHV-1 and/or EHV-4 when administered at least in part intranasally.
Throughout this application, the text refers to various embodiments of the present compositions and/or related methods. The various embodiments described are meant to provide a variety of illustrative examples and should not be construed as descriptions of alternative species. Rather, it should be noted that the descriptions of various embodiments provided herein may be of overlapping scope. The embodiments discussed herein are merely illustrative and are not meant to limit the scope of the present invention.
The present immunogenic compositions include an inactivated form of EHV-1. The vaccines are designed for protecting horses against diseases associated with EHV-1 and/or EHV-4. The vaccines typically include a chemically inactivated form of EHV-1 and those which include chemically inactivated EHV-1 KyA virus are particularly desirable. A variety of chemical inactivating agents known to those skilled in the art may be employed to inactivate the virus. Ethylenimine and related derivatives, such as binary ethylenimine (xe2x80x9cBEIxe2x80x9d) and acetylethylenimine, are examples of suitable chemical inactivating agents for use in inactivating the EHV-1 virus. Other chemical inactivating agents, e.g., beta-propiolactone, aldehydes (such as formaldehyde) and/or detergents (e.g., Tween(copyright) detergent, Triton(copyright) X, or alkyl trimethylammonium salts) can also be used to inactivate the virus. The inactivation can be performed using standard methods known to those of skill in the art. Samples can be taken at periodic time intervals and assayed for residual live virus. Monitoring of cytopathic effect on an appropriate cell line and/or fluorescent staining with an appropriate specific monoclonal antibody can be used to detected the presence of residual live virus.
Inactivation with BEI can be accomplished by combining a stock BEI solution (e.g., a solution formed by adding 0.1-0.2 M 2-bromo-ethylamine hydrobromide to 0.1-0.2 N aqueous NaOH) with viral fluids to a final concentration of about 1-2 mM BEI. Inactivation is commonly performed by holding the BEI-virus mixture at 35-40xc2x0 C. (e.g., 37xc2x0 C.) with constant mixing for 36-72 hours. Virus inactivation can be halted by the addition of sodium thiosulfate solution to a final concentration in excess of the BEI concentration (e.g., 2-3 mM sodium thiosulfate with 1-2 mM BEI solutions) followed by mixing for several hours.
The present immunogenic compositions usually include an adjuvant and, if desired, one or more emulsifiers such as Tween(copyright) detergent incorporated with the inactivated EHV-1. Suitable adjuvants include, for example, vitamin E acetate solubilisate, aluminium hydroxide, aluminium phosphate or aluminium oxide, (mineral) oil emulsions, non-ionic detergents, squalene and saponins. Other adjuvants which may be used include an oil based adjuvants such as Freund""s complete adjuvant (FCA), and Freund""s incomplete adjuvant (FIA). It has been found that cross-linked olefinically unsaturated carboxylic acid polymers, such as Carbopol(copyright) 971 polymer, are particularly suitable adjuvants for use in the present inactivated EHV-1 immunogenic compositions.
One example of such an adjuvant is an olefinically unsaturated carboxylic acid polymer produced by reaction of a monomer mixture which includes one or more olefinically unsaturated carboxylic acid monomers (such as acrylic acid and/or methacrylic acid) and a cross-linking agent. Typically, at least about 90 wt. % of the monomer mixture is olefinically unsaturated carboxylic monomer. The resulting polymer product desirably contains no more than about 0.5 wt. % and, preferably, no more than about 0.2 wt. % unreacted olefinically unsaturated carboxylic monomer. The polymerization reaction can be carried out by reaction of the monomer mixture in the presence of solvent which includes aliphatic ketone, alkyl ester or a mixture thereof. Suitable aliphatic ketones include those having 3 to 6 carbon atoms, such as acetone and cyclohexanone (as used herein the term xe2x80x9caliphatic ketonexe2x80x9d includes cycloaliphatic ketones). Examples of suitable alkyl esters include those having 3 to 6 carbon atoms, such as ethyl acetate, ethyl formate, isopropyl acetate, n-propyl acetate, butyl acetate or a mixture thereof.
Suitable olefinically unsaturated carboxylic acid polymer adjuvants desirably have a Brookfield viscosity of no more than about 40,000 cPs (at 20 rpm as a 0.5 wt. % aqueous solution at pH 7.5). Particularly suitable examples include olefinically unsaturated carboxylic acid polymers with a viscosity of no more than about 15,000 cPs and more desirably about 4,000-11,000 cPs (at 20 rpm as a 0.5 wt. % aqueous solution at pH 7.5).
One example of a suitable adjuvant includes a cross-linked acrylic acid polymer formed from a monomer mixture which includes acrylic acid and a cross-linking agent. The cross-linking agent may include a polyalkenyl polyether cross-linking agent, such as a divinyl glycol. Examples of suitable divinyl alcohols include allyl sucrose, allyl pentaerythritol, polyalkylene diol diallyl ether having a molecular weight of no more than 1000, trimethylolpropane diallyl ether, and mixtures thereof. Examples of other useful cross-linking agents are divinylbenzene, N,N-diallylacrylamide, 3,4-dihydroxy-1,5-hexadiene, 2,5-dimethyl-1,5-hexadiene and the like.
Where the vaccine is to be administered intranasally, it may be advantageous to use an adjuvant is bioadhesive with respect to mucous membranes. Bioadhesive polymers generally have the property of being able to adhere to a mucous membrane in the eyes, nose, mouth, gastrointestinal tract, vaginal cavity and rectal canal. Bioadhesive may be broadly defined as a material that adheres to a live or freshly killed biological surface such as mucus membrane or skin tissue. Bioadhesion as that phrase is used herein to define a useful bioadhesive is assayed by a procedure that measures the force required to separate two layers of freshly excised rabbit stomach tissue that are adhered together by an adhesive. Using this procedure, a bioadhesive may be defined as a material that requires a force of at least about 50 dynes/cm.sup.2 to separate two adhered, freshly excised pieces of rabbit stomach tissue, following the procedure described in U.S. Pat. No. 4,615,697, the disclosure of which is herein incorporated by reference. The upper limits for forces required to separate the freshly excised rabbit tissue are not precisely known, but are believed to be at least about 2000 dynes/cm2.
Suitable examples of adjuvants include cross-linked olefinically unsaturated carboxylic acid polymers with bioadhesive properties (e.g., Carbopol(copyright) 971 polymer, a cross-linked acrylic acid polymer available from B. F. Goodrich Co., Cleveland, Ohio). Polyacrylic acids of this type are generally crosslinked carboxy-functional polymers that contain specified amounts of carboxyl functionality and crosslinking agent. Such polymers can be a bioadhesive such that the polymers exhibit an adhesion between two pieces of freshly excised rabbit stomach tissue of at least 50 dynes/cm.sup.2 (when measured in the manner described in U.S. Pat. No. 4,615,697).
It is generally advantageous to formulate the present compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to the treated; each unit containing a predetermined quantity of the active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of inactivated EHV-1 (as well as inactivated EHV-4 and/or inactivated EIV) are dictated by and depend on among other factors (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved; (b) the limitations inherent in the art of compounding such active material for the treatment of disease; and (c) the manner of intended administration of the dosage unit form.
The principal active ingredient is typically compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as disclosed herein. A unit dosage form can, for example, contain the EHV-1 antigen in amounts ranging from 1 to about 5 relative potency units (xe2x80x9cRPUsxe2x80x9d). This amount of the antigen is generally present in from about 1 to about 25/ml of carrier. In the case of compositions containing supplementary active ingredients (e.g., inactivated EIV and/or inactivated EHV-4), the dosages are determined by reference to the usual dose and manner of administration of the supplementary active ingredients.
The present vaccines typically include inactivated EHV-1 formulated with a pharmaceutically acceptable carrier. The pharmaceutical forms suitable for injectable use commonly include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The formulation should desirably be sterile and fluid to the extent that easy syringability exists. The dosage form should be stable under the conditions of manufacture and storage and typically is preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. One possible carrier is a physiological salt solution. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal (sodium ethylmercuri-thiosalicylate), deomycin, gentamicin and the like. In many cases it may be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions, if desired, can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum, monostearate and gelatin.
Sterile injectable solutions may be prepared by incorporating the inactivated virus in the desired amount in an appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the various active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
It may also be advantageous to add a stabilizer to the present compositions to improve the stability of inactivated virus. Suitable stabilizers include, for example, glycerol/EDTA, carbohydrates (such as sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose), proteins (such as albumin or casein) and protein degradation products (e.g., partially hydrolyzed gelatin). If desired, the formulation may be buffered by methods known in the art, using reagents such as alkali metal phosphates, e.g., sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate and/or potassium dihydrogen phosphate. Other solvents, such as ethanol or propylene glycol, can be used to increase solubility of ingredients in the vaccine formulation and/or the stability of the solution. Further additives which can be used in the present formulation include conventional antioxidants and conventional chelating agents, such as ethylenediamine tetraacetic acid (EDTA).
The compositions and methods of the present invention may be illustrated by the following examples, which are presented to illustrate the present invention and to assist in teaching one of ordinary skill how to make and use the same. These examples are not intended in any way to narrow or otherwise limit the scope of the present invention.